Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins.

During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.     

Acid binding and detritylation during oligonucleotide synthesis.

Under the conditions normally used for detritylation in oligonucleotide synthesis, the haloacetic acid binds strongly to the oligonucleotide. Acetonitrile also forms a complex with the deblocking acid, in competition with the oligonucleotide, and drastically slows detritylation. Incomplete removal of acetonitrile during the deblock step may slow the kinetics enough to result in incomplete detritylation of the oligonucleotide. Acid binding to the growing oligonucleotide causes striking chromatographic effects in the presence of high oligonucleotide mass densities. In packed-bed column reactors, at low linear velocities, the acid binding almost completely depletes free acid from the deblocking solution. This results in an advancing zone within which the oligonucleotide is saturated with acid. Detritylation occurs mostly in a narrow band at the front of the advancing saturated zone. Increasing the DCA concentration in order to achieve quick saturation can give faster and more complete detritylation while minimizing the exposure time of the oligonucleotide to acid.     

Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.     

Sequence-independent and linear variation of oligonucleotide DNA binding stabilities.

The ability to equalize the DNA binding stability of comprehensive sets of oligonucleotides is imperative for the application of sequencing by hybridization technology. By substitution of ribonucleotides into an oligonucleotide composed of deoxyribonucleotides, and vice versa, the duplex stability of the oligonucleotide is changed linearly with the number of serial alternations of sugar configurations within the molecule. Since this effort occurs independently of the actual base sequence, any set of oligonucleotides could be adjusted to a defined level of binding stability.     

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.     

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.     

Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis.

Fully protected CPG-immobilized monomer, dimer and trimer oligonucleotides were used to study depurination during the chemical synthesis of oligonucleotides. Disappearance of the oligonucleotide during acid exposure time relative to an internal thymidine standard not subject to depurination was monitored by reverse phase HPLC analysis. Depurination half-times obtained for dichloroacetic acid (DCA) and trichloroacetic acid (TCA) in methylene chloride were found to be 3% DCA >> 15% DCA > 3% TCA. In order to understand the implications of depurination during DNA synthesis, the detritylation kinetics of model compounds DMT-dG-pT dimer and DMT-[17mer] mixed-base sequence were also measured. These results improve our ability to properly balance the contradictory goals of obtaining maximum detritylation with minimum depurination in oligonucleotide synthesis.     

Enhanced oligonucleotide binding to self-assembled nanofibers

A peptide nucleic acid/peptide amphiphile conjugate (PNA-PA) that self-assembles into fiber-shaped nanostructures was designed to bind oligonucleotides with high affinity and specificity. Oligonucleotide binding to PNA-PA nanofibers was studied using fluorescence polarization, and thermal stability was examined by UV-vis measurement of duplex melting temperatures. The self-assembled PNA-PA DNA system was observed to bind more strongly than the corresponding DNA-DNA duplex. We also observed single base specificity with a 16 degrees C in thermal stability. As expected from the previous PNA studies, PNA-PA RNA binding is also stronger than the corresponding RNA-RNA duplex.     

Enhanced cellular binding of concatemeric oligonucleotide complexes

Interaction of oligonucleotides condensed into long concatemeric complexes with cancer cells was investigated. Pairs of 24- and 25-mer oligodeoxyribonucleotides were designed so that they could hybridize and form concatemeric structures. Pre-assembling of the oligonucleotides into concatemers considerably enhanced their ability to bind to human embryo kidney 293 cells and neuroblastoma IMR-32 cells as compared to free oligonucleotides. Efficiency of concatemers binding to the cells is improved with increase of the length and concentration of concatemeric complexes. The obtained results suggest incorporation of pharmacologically active oligonucleotides into concatemeric complexes as an approach to improvement of their cellular interaction.     

H-DNA formation by the coding repeat elements of neisserial opa genes

The coding repeat region of opa genes from Neisseria gonorrhoeae and Neisseria meningitidis determines the expression state of their respective genes through high-frequency addition of deletion of pentanucleotide coding repeat units (CRs; CTTCT). In vitro analyses of cloned opa gene CR regions using single-strand specific nucleases, oligonucleotide protection experiments, and modifications of non-B-DNA residues indicate that the regions form structures resembling H-DNA under acidic conditions in the presence of negative supercoiling. The purine/pyrimidine strand bias and H-palindromic nature of the repeat region are consistent with sequence requirements for H-DNA formation. Sequences flanking the repeat elements are required to form the H-DNA structure in vitro as judged by the pattern of exposed non-B-DNA residues in CR sequences synthesized as oligonucleotides to form beta-galactosidase::CR translational fusions. The fusions phase vary by addition and deletion of CR elements and the rate of phase variation increases upon induction of the fusion genes. The opa gene CR region is the first reported bacterial H-DNA structure and is unique in that it lies within the coding sequence for the gene.     

Enhanced cellular binding of concatemeric oligonucleotide complexes

Interaction of oligonucleotides condensed into long concatemeric complexes with cancer cells was investigated. Pairs of 24- and 25-mer oligodeoxyribonucleotides were designed so that they could hybridize and form concatemeric structures. Pre-assembling of the oligonucleotides into concatemers considerably enhanced their ability to bind to human embryo kidney 293 cells and neuroblastoma IMR-32 cells as compared to free oligonucleotides. Efficiency of concatemers binding to the cells is improved with increase of the length and concentration of concatemeric complexes. The obtained results suggest incorporation of pharmacologically active oligonucleotides into concatemeric complexes as an approach to improvement of their cellular interaction.     

Kinetic determination of talin-actin binding

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.     

DNA-sequence and metal-ion specificity of the formation of *H-DNA.

The homopyrimidine-homopurine sequence d(CT/GA)22 undergoes, in the presence of zinc ions, transition to an altered DNA conformation (*H-DNA) which is neither H-DNA nor B-DNA. *H-DNA is characterized by a peculiar chemical reactivity pattern in which most of the polypyrimidine strand is hyperreactive to osmium tetroxide and the central part of the polypurine strand is sensitive to diethylpyrocarbonate. Formation of *H-DNA is specific of metal-ion. *H-DNA is detected in the presence of Zn++, Cd++ and Mn++. The efficiency on promoting the transition is in the order of Zn++ greater than Cd++ much greater than Mn++. Formation of *H-DNA is also specific of nucleotide sequence. From all the different homopolymeric sequences tested only the d(CT/GA)22 sequence showed the zinc-induced transition to *H-DNA. These results suggest that stabilization of *H-DNA involves the formation of a specific complex between the metal-ion and the nucleotide sequence. The biological relevance of these results is discussed in view of the important role that zinc ions play on many nucleic acids processes.     

Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection.

To further define the canonical binding site for the P5-promoted Rep proteins of the adeno-associated virus, a modified random oligonucleotide selection procedure was performed, using purified recombinant Rep protein. These results may explain the effects of Rep on cellular gene expression.     

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.